The goal of this subproject, entitled Proton Transfer Dynamics in Heme-Copper Oxidases, is to understand the fundamental mechanisms of energy transduction in the heme-copper oxidases, with a particular emphasis on the role of the protein dynamics in effecting redox driven proton pumping. The heme-copper oxidases are a class of molecular machines responsible for energy transduction in aerobic respiration by the conversion of redox energy into a transmembrane proton gradient. Recent developments provide the opportunity for the full elucidation of the molecular mechanism of redox-linked proton pumping by the heme-copper oxidases. The determination of high-resolution structures for two closely related enzymes in this family provides an essential starting point for interpreting functional studies. These structures can guide the development of testable hypotheses regarding the mechanism(s) of coupling of redox energy to proton translocation. However, the structures by themselves cannot resolve this issue, for they provide only a static picture and thus no information on the intermediate states in the redox cycle and inherently lack the resolution required to determine changes in protonation states. Another key element is the availability of specific mutations in bacterial heme-copper oxidases. These mutations allow for direct evaluation of the role of particular residues in proton pumping. A final component that contributes to the current opportunity is provided by the substantial recent technical advances that have been made in the field of time-resolved vibrational spectroscopy. These advances, which were in significant part pioneered in our labs, now provide the capability of observing protonation state changes of single amino acid residues, or redox state changes in specific structures and coupled protein conformational changes, in an enzyme of the size of cytochrome c oxidase, and following the dynamics of these changes over many decades of time. Isotope edited FTIR difference techniques coupled with site directed mutagenesis provide definitive assignment of the vibrational observables to specific, functionally related structures. Vibrational spectroscopy can thus provide the detailed structural and dynamic information about redox and protonation state changes necessary to define the molecular mechanisms of redox coupled proton transport. We propose to apply equilibrium and time-resolved vibrational techniques to elucidate the structures and dynamics of the proton conduction pathways and to elucidate the mechanism of coupling the redox reactions to proton translocation. Specifically, we plan to (1) elucidate the nature of the proton conduction pathways in CcO and (2) determine the role of protein dynamics in coupling electron and proton transfer reactions in CcO, using vibrational spectroscopy.